Throughout this application, various publications are referenced in parentheses. Full citations for these publications may be found listed alphabetically near the end of the specification. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order too more fully describe the state of the art as known to those skilled therein as of the date of the invention described and claimed herein.
In vitro cytotoxicity assays can provide preliminary information about the activity of an antineoplastic agent against a particular solid tumor. These techniques are limited not only by the difficulties in obtaining fresh tumor tissue for culture, but also in culturing human cells in explant. In vivo assessment of antineoplastic activity is limited by the length of time required for a change in size in a measurable soft tissue lesion to occur, which can sometimes be weeks. Moreover, some cancers metastasize predominantly in bone, and cannot be accurately measured by computer tomography (CT) or magnetic resonance imaging (MRI). Therefore, some patients have ineffective drugs administered needlessly until clear progression is observed in a CT scan or MRI, or clinical symptoms worsen. Apoptotic changes, however, can be observed in vitro within hours of exposure of a cancer cell to antineoplastic agents. An in vivo assay of chemotherapeutic efficacy in real time would significantly contribute to patient management by providing information regarding the activity of a drug thereby optimizing or discontinuing therapy in the patient.
Changes in intracellular sodium [Nai] have been described in a variety of biological systems during normal and pathophysiological events relevant to chemotherapy, including movement through the cell cycle, apoptosis, necrosis and transformation from normal to neoplastic tissue. Sodium nuclear magnetic resonance (Na-NMR) and Na-MRI were used in assessing intracellular sodium changes in vivo and to follow the effects of chemotherapy on a tumor [Nai]. Flow cytometry, fluorescent indicators, and atomic absorption spectroscopy was used in parallel cell culture experiments to establish cell death or cellular dysfunction and changes in intracellular [Nai] during antineoplastic exposure in vitro. Detailed postmortem studies with immunohistofluorescence and culturing of tumor cells provided further confirmation of the link between cell ions and in vivo response to antineoplastics.
The ability to assess the efficacy of a particular therapy at an early stage has enormous potential utility. Hence, various recent studies have focused on this problem and examined the utility of monitoring, for example, changes in F-18 fluorodeoxyglucose (FDG) uptake as measured with PET imaging (94) and changes in cell metabolism as measured with P-31 MR spectroscopy (1, 44, 52, 75-78, 84). However, successful applications of MR imaging technique have yet to be realized. Recent advances made in MR pulse sequence strategy and the results of tissue culture experiments have independently led to selection of the Na+ nucleus as an important diagnostic target. Application of Na-MRI, as described here, weights images toward populations of sodium nuclei which are physiologically relevant to detecting tumors and monitoring their treatment.
Due to the biological importance of Na, its relative abundance, and its sensitivity, Na-NMR is a particularly useful tool for the study of physiological and pathophysiological processes. With special relevance to study of neoplasms, intracellular concentration of H+ (pH) and Na+ [Nai] is correlated with the proliferation rate of nonneoplastic and malignant cell populations (13). Increased [Nai] is presumably related to the role of Na+ influx in initiating movement through the cell cycle, and to Na+ linkage by transmembrane exchangers to both Ca++ and H+ (51, 54).
Measurement of Na content clinically has typically been done, using single quantum (SQ) NMR techniques (25, 74). A significant disadvantage of SQ NMR is the relatively larger abundance of extracellular (EC) versus intracellular (IC) [Na]. Attempts to discern [Nai] using only SQ NMR requires paramagnetic shift reagents (SRs) which have distinct disadvantages, including: toxicity, possible drug interaction, expense, and impermeability to the blood brain barrier. An alternative MR approach to measure IC Na content is based on the interaction of Na polyanions and their resultant effects on nuclear spin transitions; spin 3/2 nuclei (such as Na and K) have a nonvanishing quadrupole moment, allowing interaction with electrostatic field gradients (EFG) (30). In certain complex environments, like those occurring in the intracellular space of cells, but not in free solution (i.e. saline), multiple quantum (MQ) spin transitions occur which can be detected by specific pulse sequencesxe2x80x94multiple-quantum filters (71, 72, 20, 69, 81, 95, 45). Thus, the presence of an MQ signal can be used to identify populations of Na nuclei by their molecular environment and to detect changes in [Nai] without the use of. shift reagents (18, 20, 37, 39, 91, 92, 20b).
This invention uses sodium MRI rather than the traditional proton MRI or phosphorous MRI. Sodium MRI is currently an ignored area in the field of commercial clinical imagers. Despite its promise due to the importance of sodium in so many cellular/organ systems, Na-MRI has failed to reach its full potential due to problems with discerning intracellular from extracellular populations of sodium nuclei. Shift reagents, which can be used in some experimental systems, are prohibited clinically. This invention distinguishes a responsive population of sodium nuclei, intracellular in origin, which responds to chemotherapeutic agents. Furthermore, this experiment weights these sodium MR images to enhance the contribution of the responsive population of sodium nuclei. Since this is done, using single quantum pulse sequences, it is readily applicable in current clinical imaging systems.
This invention utilized two human tumor lines as subjects and two representative drugs as antineoplastic agents (both of significant clinical importance, having different modes of action) illustrate that the disclosed technique can detect the response to the chemotherapy administered at an equivalent human dose level.
Defining intracellular concentration in a subcellular micro environment requires precise definitions of the dimensions of the physical domain of interest, its particular cellular location, the time frame over which the measurement is made, and the definition of concentration (as distinct from activity). It has long been known that determining intracellular sodium and total sodium with measurements based on ion selective micro electrodes, fluorescent dyes, atomic absorption spectroscopy, single quantum Na NMR, multiple quantum filtered Na NMR, electron probe microanalysis or flame photometry give different values. These values vary depending on to what extent the measurement is dependent on the ability of sodium to bind to the measuring probe. Thus since biological sodium is bound and unbound to different extent, or is sequestered in subcellular compartments, free activity varies and all sodium are not counted equally. Furthermore, there is different access of probes like fluorescent dyes. Finally, the relaxation times vary dependent with subceilular and molecular domains, with varying availability of insoluble binding entities, and with local diffusion properties. The biological literature is based on such qualified and often conflicting measurements, or on precisely defined measurements with vary imprecise biological significance. The level of precision required to quantify sodium in these images whose pulse sequences are herein taught must be assessed in the context of the standards of research fields whereby such control values or changes in sodium have been measured with other techniques and given predictive values or other significance (15, 16, 48).
A novel aspect of the MRI acquisition procedure described herein is the use of two different weightings (a weighted image and a non-weighted image) and the quantitative use of these two images to numerically derive two additional images which represent average estimated spatial profiles of important pathophysiological parameters of great clinical interest. As the phantoms represent known and spatially homogeneously distributed values of the two parameters, it is possible to link the two derivative images to real units and values.
This approach for functional imaging of tumors during chemotherapy using sodium MRI uses old techniques in an unexpected way. Its high level of relevance is associated with a high level of practical and immediate outcome, since the noninvasive nature of this diagnostic method would make it readily available for pilot studies with human subjects.
The deficiencies of the prior art are substantially ameliorated in accordance with the present invention, which is, in one aspect, an optimized sodium magnetic resonance imaging technique for enhancing intracellular sodium and diagnosing of tumors and their response to chemotherapy.
The present invention provides a method for acquiring magnetic resonance data from a sample comprising (a) applying a radio frequency pulse to a sample in a magnetic field, thereby causing alignment of nuclei populations within the sample; (b) applying a radio frequency pulse at a set time interval (TI), thereby causing a measurable signal in the transverse plane; (c) suppressing a nuclei population in the sample by suitably selecting (TI) or by applying a multiple quantum filter;(d)applying image encoding for signal acquisition of the sample in (a); (e) detecting and analyzing the output signal to obtain a weighted image; (f) applying the magnetic field to the sample in (a); (g) detecting and analyzing the output signal to obtain an unweighted image; and (h) comparing the weighted and unweighted images.
In addition, the present invention provides a method of determining the effectiveness of chemotherapy comprising
(a) administering a dose of an antineoplastic agent to a subject prior to surgical removal of a cancerous tumor,
(b) applying the method for acquiring magnetic resonance data from the subject; and (c) using the data obtained by applying the method for acquiring magnetic resonance data to determine if the antineoplastic agent has altered the nuclei populations in the subject.
Further, the present invention provides a method for detecting and characterizing tumors in a subject comprising(a)applying the method the method for acquiring magnetic resonance data to the subject, and (b) using the data obtained by applying the method for acquiring magnetic resonance data to determine the nuclei populations in the subject.
Finally, the present invention provides a method for determining cell death or cellular dysfunction in a subject by (a) applying the method for acquiring magnetic resonance data to the subject; (b) using the data obtained by applying the method for acquiring magnetic resonance data to determine the nuclei populations in the subject; and (c) comparing the weighted and unweighted images.